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68 C. Sivapathasuntharam et al. / Neurobiology of Aging 52 (2017) 66e70 Fig. 2. COX immunohistochemistry in experimental and untreated mice at 7 m (A and B). Higher levels were found in experimental animals compared to controls (C). Similar data at 12 m (D and E), which were again significantly different with greater levels in 670 nm exposed mice (F). *p < 0.05 and **p < 0.01. Abbreviations: IHC-Fr, immunohistochemistry frozen section; ONL, outer nuclear layer; OPL, outer plexiform layer; PR, photoreceptor inner segments. were similar to that in Begum et al. (2013) with background room lighting being approximately 2.256 102 W/m2. 2.2. Electrophysiology Dark-adapted mice underwent full field scotopic (intensity sequence) and photopic (single flash, intensity sequence) ERG recordings (Diagnosys LLC, Cambridge, UK) after being anesthetized intraperitoneally with 6% ketamine (National Veterinary Services Ltd, UK), 10% Dormitor (National Veterinary Services Ltd, UK), and 84% sterile water at 5 mL/g. Pupils were dilated (1%Tropicamide, Bausch and Lomb, France), and the cornea lubricated with Visco- tears (Novartis, Switzerland). Ground and reference electrodes were subdermal. ERGs were undertaken at increasing stimulus strengths using a 6500K white light at 2.5 105, 1.25 104, 1.14 103, 0.03, 0.32, 3.11, and 31.90 sct cd s m2. After the scotopic series, mice were adapted to a 20 cd m2, rod saturating back- ground for 15 minutes. Photopic responses to single white light flash stimuli of 0.3, 2.8, 28.1, and 84.2 cd s m2 were recorded with a background light of 20 cd m2. 2.3. Immunohistochemistry Mice were killed by cervical dislocation and eyes placed in 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4 for 1 hour, cryopreserved in 30% sucrose and embedded in optimum mounting medium (Agar Scientific Ltd) and cryosectioned at 10 mm. Sections were incubated for 1 hour in 5% normal donkey serum (NDS) in 0.3% Triton X-100 in PBS, then incubated overnight with COX III (goat polyclonal 1:250) diluted in 1% NDS in 0.3% Triton X- 100 in PBS. Negative controls had the primary antibody omitted. Sections were incubated for 1 hour in donkey anti-goat secondary antibody conjugated with Alexa Fluor 568 (1:2000, Invitrogen) diluted in 2% NDS in 0.3% Triton X-100 in PBS. The slides were = mounted with Vectashield (VECTOR laboratories). Photographs of sections were taken at 400 JPEG format and analyzed using Adobe Photoshop CS4 in the same manner as Begum et al. (2013). Statistical analysis was undertaken with a 2-way analysis of variance for electrophysiological data between groups over pro- gressive intensities and between groups at specific intensities (1-way analysis of variance). Statistics for the immunohistochem- istry were undertaken with a 1-tailed Mann-Whitney U test. 3. Results There are reductions in the a- and b-waves of the ERG and delays in timing with age (Birch and Anderson, 1992; Kolesnikov et al., 2010; Neveu et al., 2011). Fig. 1A shows example ERG traces from each of the groups of mice at increasing light intensities. Data from 2-month animals are represented twice alongside both 7- and 12- month animals for direct comparison. These show age-related re- ductions in amplitude and improvements with 670 nm treatment. Fig. 1B shows that significant age-related differences are present in scotopic responses in 7- and 12-month old mice compared with 2-month old mice, with reductions in the magnitude of both negative a-wave (7 months p < 0.001, 12 months p < 0.01) and subsequent positive b-wave (7 months p < 0.001, 12 months p < 0.001). At higher intensities, reductions in the a-wave were approximately 20% in the 7-month animals and approximately 30% in the 12-month animals. b-wave reductions were approximately 40% in both groups. The photopic b-waves were reduced by approximately 25%. These differences were statistically significant (7 and 12 months both p < 0.001). Age-related timing differences were not significant. Fig. 1A and B also show differences between treated and un- treated mice. Scotopic responses in treated mice were generally increased by approximately 20% in the a-wave in both 7- and 12-month mice, which was significant (7 months p < 0.05; progressive light intensities between the 3 groups (1 way ANOVA) revealed for scotopic a-wave at 2 m versus 7 m, NS for the first 2 intensities and p < 0.05 for the final. Scotopic b- wave 2 m versus 7 m NS for the first 3 intensities and p < 0.05 for the subsequent 4. Photopic b-wave 2 m versus 7 m each NS. Scotopic a-wave 2 m versus 12 m, first 2 intensities NS, final intensity p < 0.05. Scotopic b-wave 2 m versus 12 m, first 3 intensities NS, subsequent intensities all p < 0.05. Photopic b-wave 2 m versus 12 m, first 2 intensities p < 0.05, final intensity NS.PDF Image | Aging retinal function is improved by near infrared (670 nm
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